Growth and single cell kinetics of the loricate choanoflagellate Diaphanoeca grandis

Choanoflagellates are common members of planktonic communities. Some have complex life histories that involve transitions between multiple cell stages. We have grown the loricate choanoflagellate Diaphanoeca grandis on the bacterium Pantoea sp. and integrated kinetic observations at the culture level and at the single cell level. The life history of D. grandis includes a cell division cycle with a number of recognisable cell stages. Mature, loricate D. grandis were immobile and settled on the bottom substratum. Daughter cells were ejected from the lorica 30 min. after cell division, became motile and glided on the bottom substratum until they assembled a lorica. Single cell kinetics could explain overall growth kinetics in D. grandis cultures. The specific growth rate was 0.72 day−1 during exponential growth while mature D. grandis produced daughter cells at a rate of 0.9 day−1. Daughter cells took about 1.2 h to mature. D. grandis was able to abandon and replace its lorica, an event that delayed daughter cell formation by more than 2 days. The frequency of daughter cell formation varied considerably among individuals and single cell kinetics demonstrated an extensive degree of heterogeneity in D. grandis cultures, also when growth appeared to be balanced.


= ( + )
(S1) where μ max is the maximum specific growth rate and K b is the half-saturation constant.
Inhibitory effects from high bacterial concentrations are not well understood but may e.g. be described by a sigmoidal dose-response curve = (1 − 1 1 + ( 50 − )• ) (S2) where EC 50 is the effective bacterial concentration at which the specific growth rate is reduced to half its maximal value, and the coefficient H defines the steepness of the slope of the curve.
Eqs. S1 and S2 predict that maximum specific growth rates will be obtained at indefinite or zero bacterial concentrations, respectively. A combination of the two equations will result in a kinetic model that describe the limiting as well as the inhibitory effects of bacterial concentration on the specific growth rate = ( + ) (1 − 1 1 + ( 50 − )• ) (S3) In Eq. S3 (identical to Eq. 1) should μ max be considered a theoretical parameter that in reality may be unattainable by D. grandis cultures.
Numerical solution to growth model. Numerical solutions to the structured growth model, Eqs. 2-7 are shown in Eqs. S4-S9. Cell numbers, N have been converted to cell concentrations, c by division with the liquid volume of the culture. Then, the numerical solutions to Eqs. 2-7 are Eqs. S4-S9 were solved sequentially in time intervals Δt = 0.2 h, in order to construct the growth curves in Figs. 7 and S1.  (S10) with symbols as indicated in B. Figure S4. Diaphanoeca grandis. First image of culture grown at 3,000 Pantoea sp. μL -1 recorded in the oCelloScope (Fig. 4). The image covers an area of 1,300 μm × 1,200 μm. Arrows point at parental D. grandis inoculated into the culture marked 0 as they had not yet divided (cell stages described in Fig. 2). Inset shows same individual at an expanded scale. Individual marked 0* is described in further details in Figs. S7-S9 and S11-S12. Figure S5. Diaphanoeca grandis. Last image of culture grown at 3,000 Pantoea sp. μL -1 recorded in the oCelloScope after 118.3 h (Fig. 4). The image covers an area of 1,300 μm × 1,200 μm. Arrows indicate the same 4 parental D. grandis as in Fig. S4. Inset shows same individuals at an expanded scale, e indicates empty lorica that have been abandoned by their protoplasts, m indicates mature, loricate D. grandis (cell stages described in Fig. 2). Individual marked e* (empty lorica) is described in further details in Figs. S7-S9 and S11-S12. Table S1. Diaphanoeca grandis. Overall data from 3 cultures grown in the oCelloScope (Fig. 4). Initial concentrations of Pantoea sp., c b,0 . total number of D. grandis in the filmed area at t = 0, N 0 and at t = 120 h, N 120 . Total increase in numbers of D. grandis and cell doublings during 120 h. Length of lag phase until exponential growth, and specific growth rate, μ during exponential growth phases.  Figure S7. Diaphanoeca grandis. Time series of images of dividing protoplast 2.6 days after the culture had been inoculated into the oCelloScope. The first image of mature cell, m is obtained 7 min before cells were visibly dividing, d. After 27 min the non-loricate daughter protoplast, n, abandoned its parental lorica (cell stages described in Fig. 2). Figure S8. Diaphanoeca grandis. Time series of images of daughter cell forming a lorica 2.6 days after the culture had been inoculated into the oCelloScope. The individual is the daughter cell in Fig. S7. The first image of the daughter cell, n, is obtained 50 min after the first image in Fig. S7. The daughter cell began assembling its lorica after 55 min, which after 70 min was completed, and a mature D. grandis, m, was formed (cell stages described in Fig. 2). The protoplast localizes itself differently within the lorica when it is being assembled compared to after the lorica has been completed. Arrows in the image obtained at 50 min indicate the elongated cytoplasmic strand connecting mother and daughter cell, which in this event did not break, and what presumably are costal strips carried on the surface of the motile, non-loricate daughter cell. The daughter cell settled with a distance less than 50 μm from the parental lorica, although it travelled more than 3 times this distance (Fig. S9C). The sequence is also shown in Supplementary Video S1. Figure S9. Diaphanoeca grandis. Examples of trajectories (A and C, parental lorica is positioned in (0, 0)) and velocities (B and D) of motile, non-loricate daughter cells, in one event where the elongated cytoplasmic strand came apart and the daughter cell was released (A and B, total distance travelled = 480 μm) and in one event where the elongated cytoplasmic strand did not break (C and D, same daughter cell as in Figs. S7 and S8, total distance travelled = 183 μm). The following events have been indicated by numbers: 1, two cells visible within parental lorica. 2, non-loricate daughter protoplast leaves parental lorica. 3, elongated cytoplasmic strand breaks. 4, daughter cell starts assembling its new lorica. 5, the new lorica is completed. Figure S10. Diaphanoeca grandis, total number of daughter cells produced by individual D. grandis inoculated and filmed in the oCelloScope and grown on 3,000 (N 0 = 38, A), 6,000 (N 0 = 40, B) and 12,000 (N 0 = 40,C) Pantoea sp. μL -1 (Figs. 4-6). Figure S11. Diaphanoeca grandis. Time series of images of protoplast of mature D. grandis, m, abandoning its lorica 3.8 days after the culture had been inoculated into the oCelloScope. The individual is the same as in Fig. S7. The non-loricate protoplast, n, abandoned and left behind the empty lorica, e, 26 min into the sequence. It began to assemble a new lorica, l, 59 min into the sequence. After 70 min the new lorica was completed, and a mature D. grandis, m, was again formed (cell stages described in Fig. 2). The motile, non-loricate cell dragged behind itself an elongated cytoplasmic strand although there was no mother cell to connect to, and it carried what appeared to be costal strips, both structures indicated by arrows in the image taken 38 min into the sequence. The sequence is also shown in Supplementary Video S1.  Supplementary Video S1. Diaphanoeca grandis. Dividing protoplast 2.6 days after the culture had been inoculated into the oCelloScope. Same parental cell indicated by arrow as in Figs. S7, and same daughter cell as in Fig. S8. The video also shows additional mature D. grandis, dividing protoplast, and motile, non-loricate cells. Total length of video corresponds to 100 min.
Supplementary Video S2. Diaphanoeca grandis. Mature protoplast abandoning its lorica 3.8 days after the culture had been inoculated into the oCelloScope. Same parental cell indicated by arrow as in Fig. S11. The renewed lorica, also shown in Fig. S11 is also indicated by arrow. The video also shows additional mature D. grandis, dividing protoplast, motile, non-loricate cells, and cells forming a lorica. Total length of video corresponds to 100 min.